cloning and stem cell technology essay

• Open access
• Published: 26 February 2019

Stem cells: past, present, and future

• Wojciech Zakrzewski 1 ,
• Maciej Dobrzyński 2 ,
• Maria Szymonowicz 1 &
• Zbigniew Rybak 1  

Stem Cell Research & Therapy volume  10 , Article number:  68 ( 2019 ) Cite this article

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In recent years, stem cell therapy has become a very promising and advanced scientific research topic. The development of treatment methods has evoked great expectations. This paper is a review focused on the discovery of different stem cells and the potential therapies based on these cells. The genesis of stem cells is followed by laboratory steps of controlled stem cell culturing and derivation. Quality control and teratoma formation assays are important procedures in assessing the properties of the stem cells tested. Derivation methods and the utilization of culturing media are crucial to set proper environmental conditions for controlled differentiation. Among many types of stem tissue applications, the use of graphene scaffolds and the potential of extracellular vesicle-based therapies require attention due to their versatility. The review is summarized by challenges that stem cell therapy must overcome to be accepted worldwide. A wide variety of possibilities makes this cutting edge therapy a turning point in modern medicine, providing hope for untreatable diseases.

Stem cell classification

Stem cells are unspecialized cells of the human body. They are able to differentiate into any cell of an organism and have the ability of self-renewal. Stem cells exist both in embryos and adult cells. There are several steps of specialization. Developmental potency is reduced with each step, which means that a unipotent stem cell is not able to differentiate into as many types of cells as a pluripotent one. This chapter will focus on stem cell classification to make it easier for the reader to comprehend the following chapters.

Totipotent stem cells are able to divide and differentiate into cells of the whole organism. Totipotency has the highest differentiation potential and allows cells to form both embryo and extra-embryonic structures. One example of a totipotent cell is a zygote, which is formed after a sperm fertilizes an egg. These cells can later develop either into any of the three germ layers or form a placenta. After approximately 4 days, the blastocyst’s inner cell mass becomes pluripotent. This structure is the source of pluripotent cells.

Pluripotent stem cells (PSCs) form cells of all germ layers but not extraembryonic structures, such as the placenta. Embryonic stem cells (ESCs) are an example. ESCs are derived from the inner cell mass of preimplantation embryos. Another example is induced pluripotent stem cells (iPSCs) derived from the epiblast layer of implanted embryos. Their pluripotency is a continuum, starting from completely pluripotent cells such as ESCs and iPSCs and ending on representatives with less potency—multi-, oligo- or unipotent cells. One of the methods to assess their activity and spectrum is the teratoma formation assay. iPSCs are artificially generated from somatic cells, and they function similarly to PSCs. Their culturing and utilization are very promising for present and future regenerative medicine.

Multipotent stem cells have a narrower spectrum of differentiation than PSCs, but they can specialize in discrete cells of specific cell lineages. One example is a haematopoietic stem cell, which can develop into several types of blood cells. After differentiation, a haematopoietic stem cell becomes an oligopotent cell. Its differentiation abilities are then restricted to cells of its lineage. However, some multipotent cells are capable of conversion into unrelated cell types, which suggests naming them pluripotent cells.

Oligopotent stem cells can differentiate into several cell types. A myeloid stem cell is an example that can divide into white blood cells but not red blood cells.

Unipotent stem cells are characterized by the narrowest differentiation capabilities and a special property of dividing repeatedly. Their latter feature makes them a promising candidate for therapeutic use in regenerative medicine. These cells are only able to form one cell type, e.g. dermatocytes.

Stem cell biology

A blastocyst is formed after the fusion of sperm and ovum fertilization. Its inner wall is lined with short-lived stem cells, namely, embryonic stem cells. Blastocysts are composed of two distinct cell types: the inner cell mass (ICM), which develops into epiblasts and induces the development of a foetus, and the trophectoderm (TE). Blastocysts are responsible for the regulation of the ICM microenvironment. The TE continues to develop and forms the extraembryonic support structures needed for the successful origin of the embryo, such as the placenta. As the TE begins to form a specialized support structure, the ICM cells remain undifferentiated, fully pluripotent and proliferative [ 1 ]. The pluripotency of stem cells allows them to form any cell of the organism. Human embryonic stem cells (hESCs) are derived from the ICM. During the process of embryogenesis, cells form aggregations called germ layers: endoderm, mesoderm and ectoderm (Fig.  1 ), each eventually giving rise to differentiated cells and tissues of the foetus and, later on, the adult organism [ 2 ]. After hESCs differentiate into one of the germ layers, they become multipotent stem cells, whose potency is limited to only the cells of the germ layer. This process is short in human development. After that, pluripotent stem cells occur all over the organism as undifferentiated cells, and their key abilities are proliferation by the formation of the next generation of stem cells and differentiation into specialized cells under certain physiological conditions.

Oocyte development and formation of stem cells: the blastocoel, which is formed from oocytes, consists of embryonic stem cells that later differentiate into mesodermal, ectodermal, or endodermal cells. Blastocoel develops into the gastrula

Signals that influence the stem cell specialization process can be divided into external, such as physical contact between cells or chemical secretion by surrounding tissue, and internal, which are signals controlled by genes in DNA.

Stem cells also act as internal repair systems of the body. The replenishment and formation of new cells are unlimited as long as an organism is alive. Stem cell activity depends on the organ in which they are in; for example, in bone marrow, their division is constant, although in organs such as the pancreas, division only occurs under special physiological conditions.

Stem cell functional division

Whole-body development.

During division, the presence of different stem cells depends on organism development. Somatic stem cell ESCs can be distinguished. Although the derivation of ESCs without separation from the TE is possible, such a combination has growth limits. Because proliferating actions are limited, co-culture of these is usually avoided.

ESCs are derived from the inner cell mass of the blastocyst, which is a stage of pre-implantation embryo ca. 4 days after fertilization. After that, these cells are placed in a culture dish filled with culture medium. Passage is an inefficient but popular process of sub-culturing cells to other dishes. These cells can be described as pluripotent because they are able to eventually differentiate into every cell type in the organism. Since the beginning of their studies, there have been ethical restrictions connected to the medical use of ESCs in therapies. Most embryonic stem cells are developed from eggs that have been fertilized in an in vitro clinic, not from eggs fertilized in vivo.

Somatic or adult stem cells are undifferentiated and found among differentiated cells in the whole body after development. The function of these cells is to enable the healing, growth, and replacement of cells that are lost each day. These cells have a restricted range of differentiation options. Among many types, there are the following:

Mesenchymal stem cells are present in many tissues. In bone marrow, these cells differentiate mainly into the bone, cartilage, and fat cells. As stem cells, they are an exception because they act pluripotently and can specialize in the cells of any germ layer.

Neural cells give rise to nerve cells and their supporting cells—oligodendrocytes and astrocytes.

Haematopoietic stem cells form all kinds of blood cells: red, white, and platelets.

Skin stem cells form, for example, keratinocytes, which form a protective layer of skin.

The proliferation time of somatic stem cells is longer than that of ESCs. It is possible to reprogram adult stem cells back to their pluripotent state. This can be performed by transferring the adult nucleus into the cytoplasm of an oocyte or by fusion with the pluripotent cell. The same technique was used during cloning of the famous Dolly sheep.

hESCs are involved in whole-body development. They can differentiate into pluripotent, totipotent, multipotent, and unipotent cells (Fig.  2 ) [ 2 ].

Changes in the potency of stem cells in human body development. Potency ranges from pluripotent cells of the blastocyst to unipotent cells of a specific tissue in a human body such as the skin, CNS, or bone marrow. Reversed pluripotency can be achieved by the formation of induced pluripotent stem cells using either octamer-binding transcription factor (Oct4), sex-determining region Y (Sox2), Kruppel-like factor 4 (Klf4), or the Myc gene

Pluripotent cells can be named totipotent if they can additionally form extraembryonic tissues of the embryo. Multipotent cells are restricted in differentiating to each cell type of given tissue. When tissue contains only one lineage of cells, stem cells that form them are called either called oligo- or unipotent.

iPSC quality control and recognition by morphological differences

The comparability of stem cell lines from different individuals is needed for iPSC lines to be used in therapeutics [ 3 ]. Among critical quality procedures, the following can be distinguished:

Short tandem repeat analysis—This is the comparison of specific loci on the DNA of the samples. It is used in measuring an exact number of repeating units. One unit consists of 2 to 13 nucleotides repeating many times on the DNA strand. A polymerase chain reaction is used to check the lengths of short tandem repeats. The genotyping procedure of source tissue, cells, and iPSC seed and master cell banks is recommended.

Identity analysis—The unintentional switching of lines, resulting in other stem cell line contamination, requires rigorous assay for cell line identification.

Residual vector testing—An appearance of reprogramming vectors integrated into the host genome is hazardous, and testing their presence is a mandatory procedure. It is a commonly used procedure for generating high-quality iPSC lines. An acceptable threshold in high-quality research-grade iPSC line collections is ≤ 1 plasmid copies per 100 cells. During the procedure, 2 different regions, common to all plasmids, should be used as specific targets, such as EBNA and CAG sequences [ 3 ]. To accurately represent the test reactions, a standard curve needs to be prepared in a carrier of gDNA from a well-characterized hPSC line. For calculations of plasmid copies per cell, it is crucial to incorporate internal reference gDNA sequences to allow the quantification of, for example, ribonuclease P (RNaseP) or human telomerase reverse transcriptase (hTERT).

Karyotype—A long-term culture of hESCs can accumulate culture-driven mutations [ 4 ]. Because of that, it is crucial to pay additional attention to genomic integrity. Karyotype tests can be performed by resuscitating representative aliquots and culturing them for 48–72 h before harvesting cells for karyotypic analysis. If abnormalities are found within the first 20 karyotypes, the analysis must be repeated on a fresh sample. When this situation is repeated, the line is evaluated as abnormal. Repeated abnormalities must be recorded. Although karyology is a crucial procedure in stem cell quality control, the single nucleotide polymorphism (SNP) array, discussed later, has approximately 50 times higher resolution.

Viral testing—When assessing the quality of stem cells, all tests for harmful human adventitious agents must be performed (e.g. hepatitis C or human immunodeficiency virus). This procedure must be performed in the case of non-xeno-free culture agents.

Bacteriology—Bacterial or fungal sterility tests can be divided into culture- or broth-based tests. All the procedures must be recommended by pharmacopoeia for the jurisdiction in which the work is performed.

Single nucleotide polymorphism arrays—This procedure is a type of DNA microarray that detects population polymorphisms by enabling the detection of subchromosomal changes and the copy-neutral loss of heterozygosity, as well as an indication of cellular transformation. The SNP assay consists of three components. The first is labelling fragmented nucleic acid sequences with fluorescent dyes. The second is an array that contains immobilized allele-specific oligonucleotide (ASO) probes. The last component detects, records, and eventually interprets the signal.

Flow cytometry—This is a technique that utilizes light to count and profile cells in a heterogeneous fluid mixture. It allows researchers to accurately and rapidly collect data from heterogeneous fluid mixtures with live cells. Cells are passed through a narrow channel one by one. During light illumination, sensors detect light emitted or refracted from the cells. The last step is data analysis, compilation and integration into a comprehensive picture of the sample.

Phenotypic pluripotency assays—Recognizing undifferentiated cells is crucial in successful stem cell therapy. Among other characteristics, stem cells appear to have a distinct morphology with a high nucleus to cytoplasm ratio and a prominent nucleolus. Cells appear to be flat with defined borders, in contrast to differentiating colonies, which appear as loosely located cells with rough borders [ 5 ]. It is important that images of ideal and poor quality colonies for each cell line are kept in laboratories, so whenever there is doubt about the quality of culture, it can always be checked according to the representative image. Embryoid body formation or directed differentiation of monolayer cultures to produce cell types representative of all three embryonic germ layers must be performed. It is important to note that colonies cultured under different conditions may have different morphologies [ 6 ].

Histone modification and DNA methylation—Quality control can be achieved by using epigenetic analysis tools such as histone modification or DNA methylation. When stem cells differentiate, the methylation process silences pluripotency genes, which reduces differentiation potential, although other genes may undergo demethylation to become expressed [ 7 ]. It is important to emphasize that stem cell identity, together with its morphological characteristics, is also related to its epigenetic profile [ 8 , 9 ]. According to Brindley [ 10 ], there is a relationship between epigenetic changes, pluripotency, and cell expansion conditions, which emphasizes that unmethylated regions appear to be serum-dependent.

hESC derivation and media

hESCs can be derived using a variety of methods, from classic culturing to laser-assisted methodologies or microsurgery [ 11 ]. hESC differentiation must be specified to avoid teratoma formation (see Fig.  3 ).

Spontaneous differentiation of hESCs causes the formation of a heterogeneous cell population. There is a different result, however, when commitment signals (in forms of soluble factors and culture conditions) are applied and enable the selection of progenitor cells

hESCs spontaneously differentiate into embryonic bodies (EBs) [ 12 ]. EBs can be studied instead of embryos or animals to predict their effects on early human development. There are many different methods for acquiring EBs, such as bioreactor culture [ 13 ], hanging drop culture [ 12 ], or microwell technology [ 14 , 15 ]. These methods allow specific precursors to form in vitro [ 16 ].

The essential part of these culturing procedures is a separation of inner cell mass to culture future hESCs (Fig.  4 ) [ 17 ]. Rosowski et al. [ 18 ] emphasizes that particular attention must be taken in controlling spontaneous differentiation. When the colony reaches the appropriate size, cells must be separated. The occurrence of pluripotent cells lasts for 1–2 days. Because the classical utilization of hESCs caused ethical concerns about gastrulas used during procedures, Chung et al. [ 19 ] found out that it is also possible to obtain hESCs from four cell embryos, leaving a higher probability of embryo survival. Additionally, Zhang et al. [ 20 ] used only in vitro fertilization growth-arrested cells.

Culturing of pluripotent stem cells in vitro. Three days after fertilization, totipotent cells are formed. Blastocysts with ICM are formed on the sixth day after fertilization. Pluripotent stem cells from ICM can then be successfully transmitted on a dish

Cell passaging is used to form smaller clusters of cells on a new culture surface [ 21 ]. There are four important passaging procedures.

Enzymatic dissociation is a cutting action of enzymes on proteins and adhesion domains that bind the colony. It is a gentler method than the manual passage. It is crucial to not leave hESCs alone after passaging. Solitary cells are more sensitive and can easily undergo cell death; collagenase type IV is an example [ 22 , 23 ].

Manual passage , on the other hand, focuses on using cell scratchers. The selection of certain cells is not necessary. This should be done in the early stages of cell line derivation [ 24 ].

Trypsin utilization allows a healthy, automated hESC passage. Good Manufacturing Practice (GMP)-grade recombinant trypsin is widely available in this procedure [ 24 ]. However, there is a risk of decreasing the pluripotency and viability of stem cells [ 25 ]. Trypsin utilization can be halted with an inhibitor of the protein rho-associated protein kinase (ROCK) [ 26 ].

Ethylenediaminetetraacetic acid ( EDTA ) indirectly suppresses cell-to-cell connections by chelating divalent cations. Their suppression promotes cell dissociation [ 27 ].

Stem cells require a mixture of growth factors and nutrients to differentiate and develop. The medium should be changed each day.

Traditional culture methods used for hESCs are mouse embryonic fibroblasts (MEFs) as a feeder layer and bovine serum [ 28 ] as a medium. Martin et al. [ 29 ] demonstrated that hESCs cultured in the presence of animal products express the non-human sialic acid, N -glycolylneuraminic acid (NeuGc). Feeder layers prevent uncontrolled proliferation with factors such as leukaemia inhibitory factor (LIF) [ 30 ].

First feeder layer-free culture can be supplemented with serum replacement, combined with laminin [ 31 ]. This causes stable karyotypes of stem cells and pluripotency lasting for over a year.

Initial culturing media can be serum (e.g. foetal calf serum FCS), artificial replacement such as synthetic serum substitute (SSS), knockout serum replacement (KOSR), or StemPro [ 32 ]. The simplest culture medium contains only eight essential elements: DMEM/F12 medium, selenium, NaHCO 3, l -ascorbic acid, transferrin, insulin, TGFβ1, and FGF2 [ 33 ]. It is not yet fully known whether culture systems developed for hESCs can be allowed without adaptation in iPSC cultures.

Turning point in stem cell therapy

The turning point in stem cell therapy appeared in 2006, when scientists Shinya Yamanaka, together with Kazutoshi Takahashi, discovered that it is possible to reprogram multipotent adult stem cells to the pluripotent state. This process avoided endangering the foetus’ life in the process. Retrovirus-mediated transduction of mouse fibroblasts with four transcription factors (Oct-3/4, Sox2, KLF4, and c-Myc) [ 34 ] that are mainly expressed in embryonic stem cells could induce the fibroblasts to become pluripotent (Fig.  5 ) [ 35 ]. This new form of stem cells was named iPSCs. One year later, the experiment also succeeded with human cells [ 36 ]. After this success, the method opened a new field in stem cell research with a generation of iPSC lines that can be customized and biocompatible with the patient. Recently, studies have focused on reducing carcinogenesis and improving the conduction system.

Retroviral-mediated transduction induces pluripotency in isolated patient somatic cells. Target cells lose their role as somatic cells and, once again, become pluripotent and can differentiate into any cell type of human body

The turning point was influenced by former discoveries that happened in 1962 and 1987.

The former discovery was about scientist John Gurdon successfully cloning frogs by transferring a nucleus from a frog’s somatic cells into an oocyte. This caused a complete reversion of somatic cell development [ 37 ]. The results of his experiment became an immense discovery since it was previously believed that cell differentiation is a one-way street only, but his experiment suggested the opposite and demonstrated that it is even possible for a somatic cell to again acquire pluripotency [ 38 ].

The latter was a discovery made by Davis R.L. that focused on fibroblast DNA subtraction. Three genes were found that originally appeared in myoblasts. The enforced expression of only one of the genes, named myogenic differentiation 1 (Myod1), caused the conversion of fibroblasts into myoblasts, showing that reprogramming cells is possible, and it can even be used to transform cells from one lineage to another [ 39 ].

Although pluripotency can occur naturally only in embryonic stem cells, it is possible to induce terminally differentiated cells to become pluripotent again. The process of direct reprogramming converts differentiated somatic cells into iPSC lines that can form all cell types of an organism. Reprogramming focuses on the expression of oncogenes such as Myc and Klf4 (Kruppel-like factor 4). This process is enhanced by a downregulation of genes promoting genome stability, such as p53. Additionally, cell reprogramming involves histone alteration. All these processes can cause potential mutagenic risk and later lead to an increased number of mutations. Quinlan et al. [ 40 ] checked fully pluripotent mouse iPSCs using whole genome DNA sequencing and structural variation (SV) detection algorithms. Based on those studies, it was confirmed that although there were single mutations in the non-genetic region, there were non-retrotransposon insertions. This led to the conclusion that current reprogramming methods can produce fully pluripotent iPSCs without severe genomic alterations.

During the course of development from pluripotent hESCs to differentiated somatic cells, crucial changes appear in the epigenetic structure of these cells. There is a restriction or permission of the transcription of genes relevant to each cell type. When somatic cells are being reprogrammed using transcription factors, all the epigenetic architecture has to be reconditioned to achieve iPSCs with pluripotency [ 41 ]. However, cells of each tissue undergo specific somatic genomic methylation. This influences transcription, which can further cause alterations in induced pluripotency [ 42 ].

Source of iPSCs

Because pluripotent cells can propagate indefinitely and differentiate into any kind of cell, they can be an unlimited source, either for replacing lost or diseased tissues. iPSCs bypass the need for embryos in stem cell therapy. Because they are made from the patient’s own cells, they are autologous and no longer generate any risk of immune rejection.

At first, fibroblasts were used as a source of iPSCs. Because a biopsy was needed to achieve these types of cells, the technique underwent further research. Researchers investigated whether more accessible cells could be used in the method. Further, other cells were used in the process: peripheral blood cells, keratinocytes, and renal epithelial cells found in urine. An alternative strategy to stem cell transplantation can be stimulating a patient’s endogenous stem cells to divide or differentiate, occurring naturally when skin wounds are healing. In 2008, pancreatic exocrine cells were shown to be reprogrammed to functional, insulin-producing beta cells [ 43 ].

The best stem cell source appears to be the fibroblasts, which is more tempting in the case of logistics since its stimulation can be fast and better controlled [ 44 ].

• Teratoma formation assay

The self-renewal and differentiation capabilities of iPSCs have gained significant interest and attention in regenerative medicine sciences. To study their abilities, a quality-control assay is needed, of which one of the most important is the teratoma formation assay. Teratomas are benign tumours. Teratomas are capable of rapid growth in vivo and are characteristic because of their ability to develop into tissues of all three germ layers simultaneously. Because of the high pluripotency of teratomas, this formation assay is considered an assessment of iPSC’s abilities [ 45 ].

Teratoma formation rate, for instance, was observed to be elevated in human iPSCs compared to that in hESCs [ 46 ]. This difference may be connected to different differentiation methods and cell origins. Most commonly, the teratoma assay involves an injection of examined iPSCs subcutaneously or under the testis or kidney capsule in mice, which are immune-deficient [ 47 ]. After injection, an immature but recognizable tissue can be observed, such as the kidney tubules, bone, cartilage, or neuroepithelium [ 30 ]. The injection site may have an impact on the efficiency of teratoma formation [ 48 ].

There are three groups of markers used in this assay to differentiate the cells of germ layers. For endodermal tissue, there is insulin/C-peptide and alpha-1 antitrypsin [ 49 ]. For the mesoderm, derivatives can be used, e.g. cartilage matrix protein for the bone and alcian blue for the cartilage. As ectodermal markers, class III B botulin or keratin can be used for keratinocytes.

Teratoma formation assays are considered the gold standard for demonstrating the pluripotency of human iPSCs, demonstrating their possibilities under physiological conditions. Due to their actual tissue formation, they could be used for the characterization of many cell lineages [ 50 ].

Directed differentiation

To be useful in therapy, stem cells must be converted into desired cell types as necessary or else the whole regenerative medicine process will be pointless. Differentiation of ESCs is crucial because undifferentiated ESCs can cause teratoma formation in vivo. Understanding and using signalling pathways for differentiation is an important method in successful regenerative medicine. In directed differentiation, it is likely to mimic signals that are received by cells when they undergo successive stages of development [ 51 ]. The extracellular microenvironment plays a significant role in controlling cell behaviour. By manipulating the culture conditions, it is possible to restrict specific differentiation pathways and generate cultures that are enriched in certain precursors in vitro. However, achieving a similar effect in vivo is challenging. It is crucial to develop culture conditions that will allow the promotion of homogenous and enhanced differentiation of ESCs into functional and desired tissues.

Regarding the self-renewal of embryonic stem cells, Hwang et al. [ 52 ] noted that the ideal culture method for hESC-based cell and tissue therapy would be a defined culture free of either the feeder layer or animal components. This is because cell and tissue therapy requires the maintenance of large quantities of undifferentiated hESCs, which does not make feeder cells suitable for such tasks.

Most directed differentiation protocols are formed to mimic the development of an inner cell mass during gastrulation. During this process, pluripotent stem cells differentiate into ectodermal, mesodermal, or endodermal progenitors. Mall molecules or growth factors induce the conversion of stem cells into appropriate progenitor cells, which will later give rise to the desired cell type. There is a variety of signal intensities and molecular families that may affect the establishment of germ layers in vivo, such as fibroblast growth factors (FGFs) [ 53 ]; the Wnt family [ 54 ] or superfamily of transforming growth factors—β(TGFβ); and bone morphogenic proteins (BMP) [ 55 ]. Each candidate factor must be tested on various concentrations and additionally applied to various durations because the precise concentrations and times during which developing cells in embryos are influenced during differentiation are unknown. For instance, molecular antagonists of endogenous BMP and Wnt signalling can be used for ESC formation of ectoderm [ 56 ]. However, transient Wnt and lower concentrations of the TGFβ family trigger mesodermal differentiation [ 57 ]. Regarding endoderm formation, a higher activin A concentration may be required [ 58 , 59 ].

There are numerous protocols about the methods of forming progenitors of cells of each of germ layers, such as cardiomyocytes [ 60 ], hepatocytes [ 61 ], renal cells [ 62 ], lung cells [ 63 , 64 ], motor neurons [ 65 ], intestinal cells [ 66 ], or chondrocytes [ 67 ].

Directed differentiation of either iPSCs or ESCs into, e.g. hepatocytes, could influence and develop the study of the molecular mechanisms in human liver development. In addition, it could also provide the possibility to form exogenous hepatocytes for drug toxicity testing [ 68 ].

Levels of concentration and duration of action with a specific signalling molecule can cause a variety of factors. Unfortunately, for now, a high cost of recombinant factors is likely to limit their use on a larger scale in medicine. The more promising technique focuses on the use of small molecules. These can be used for either activating or deactivating specific signalling pathways. They enhance reprogramming efficiency by creating cells that are compatible with the desired type of tissue. It is a cheaper and non-immunogenic method.

One of the successful examples of small-molecule cell therapies is antagonists and agonists of the Hedgehog pathway. They show to be very useful in motor neuron regeneration [ 69 ]. Endogenous small molecules with their function in embryonic development can also be used in in vitro methods to induce the differentiation of cells; for example, retinoic acid, which is responsible for patterning the nervous system in vivo [ 70 ], surprisingly induced retinal cell formation when the laboratory procedure involved hESCs [ 71 ].

The efficacy of differentiation factors depends on functional maturity, efficiency, and, finally, introducing produced cells to their in vivo equivalent. Topography, shear stress, and substrate rigidity are factors influencing the phenotype of future cells [ 72 ].

The control of biophysical and biochemical signals, the biophysical environment, and a proper guide of hESC differentiation are important factors in appropriately cultured stem cells.

Stem cell utilization and their manufacturing standards and culture systems

The European Medicines Agency and the Food and Drug Administration have set Good Manufacturing Practice (GMP) guidelines for safe and appropriate stem cell transplantation. In the past, protocols used for stem cell transplantation required animal-derived products [ 73 ].

The risk of introducing animal antigens or pathogens caused a restriction in their use. Due to such limitations, the technique required an obvious update [ 74 ]. Now, it is essential to use xeno-free equivalents when establishing cell lines that are derived from fresh embryos and cultured from human feeder cell lines [ 75 ]. In this method, it is crucial to replace any non-human materials with xeno-free equivalents [ 76 ].

NutriStem with LN-511, TeSR2 with human recombinant laminin (LN-511), and RegES with human foreskin fibroblasts (HFFs) are commonly used xeno-free culture systems [ 33 ]. There are many organizations and international initiatives, such as the National Stem Cell Bank, that provide stem cell lines for treatment or medical research [ 77 ].

Stem cell use in medicine

Stem cells have great potential to become one of the most important aspects of medicine. In addition to the fact that they play a large role in developing restorative medicine, their study reveals much information about the complex events that happen during human development.

The difference between a stem cell and a differentiated cell is reflected in the cells’ DNA. In the former cell, DNA is arranged loosely with working genes. When signals enter the cell and the differentiation process begins, genes that are no longer needed are shut down, but genes required for the specialized function will remain active. This process can be reversed, and it is known that such pluripotency can be achieved by interaction in gene sequences. Takahashi and Yamanaka [ 78 ] and Loh et al. [ 79 ] discovered that octamer-binding transcription factor 3 and 4 (Oct3/4), sex determining region Y (SRY)-box 2 and Nanog genes function as core transcription factors in maintaining pluripotency. Among them, Oct3/4 and Sox2 are essential for the generation of iPSCs.

Many serious medical conditions, such as birth defects or cancer, are caused by improper differentiation or cell division. Currently, several stem cell therapies are possible, among which are treatments for spinal cord injury, heart failure [ 80 ], retinal and macular degeneration [ 81 ], tendon ruptures, and diabetes type 1 [ 82 ]. Stem cell research can further help in better understanding stem cell physiology. This may result in finding new ways of treating currently incurable diseases.

Haematopoietic stem cell transplantation

Haematopoietic stem cells are important because they are by far the most thoroughly characterized tissue-specific stem cell; after all, they have been experimentally studied for more than 50 years. These stem cells appear to provide an accurate paradigm model system to study tissue-specific stem cells, and they have potential in regenerative medicine.

Multipotent haematopoietic stem cell (HSC) transplantation is currently the most popular stem cell therapy. Target cells are usually derived from the bone marrow, peripheral blood, or umbilical cord blood [ 83 ]. The procedure can be autologous (when the patient’s own cells are used), allogenic (when the stem cell comes from a donor), or syngeneic (from an identical twin). HSCs are responsible for the generation of all functional haematopoietic lineages in blood, including erythrocytes, leukocytes, and platelets. HSC transplantation solves problems that are caused by inappropriate functioning of the haematopoietic system, which includes diseases such as leukaemia and anaemia. However, when conventional sources of HSC are taken into consideration, there are some important limitations. First, there is a limited number of transplantable cells, and an efficient way of gathering them has not yet been found. There is also a problem with finding a fitting antigen-matched donor for transplantation, and viral contamination or any immunoreactions also cause a reduction in efficiency in conventional HSC transplantations. Haematopoietic transplantation should be reserved for patients with life-threatening diseases because it has a multifactorial character and can be a dangerous procedure. iPSC use is crucial in this procedure. The use of a patient’s own unspecialized somatic cells as stem cells provides the greatest immunological compatibility and significantly increases the success of the procedure.

Stem cells as a target for pharmacological testing

Stem cells can be used in new drug tests. Each experiment on living tissue can be performed safely on specific differentiated cells from pluripotent cells. If any undesirable effect appears, drug formulas can be changed until they reach a sufficient level of effectiveness. The drug can enter the pharmacological market without harming any live testers. However, to test the drugs properly, the conditions must be equal when comparing the effects of two drugs. To achieve this goal, researchers need to gain full control of the differentiation process to generate pure populations of differentiated cells.

Stem cells as an alternative for arthroplasty

One of the biggest fears of professional sportsmen is getting an injury, which most often signifies the end of their professional career. This applies especially to tendon injuries, which, due to current treatment options focusing either on conservative or surgical treatment, often do not provide acceptable outcomes. Problems with the tendons start with their regeneration capabilities. Instead of functionally regenerating after an injury, tendons merely heal by forming scar tissues that lack the functionality of healthy tissues. Factors that may cause this failed healing response include hypervascularization, deposition of calcific materials, pain, or swelling [ 84 ].

Additionally, in addition to problems with tendons, there is a high probability of acquiring a pathological condition of joints called osteoarthritis (OA) [ 85 ]. OA is common due to the avascular nature of articular cartilage and its low regenerative capabilities [ 86 ]. Although arthroplasty is currently a common procedure in treating OA, it is not ideal for younger patients because they can outlive the implant and will require several surgical procedures in the future. These are situations where stem cell therapy can help by stopping the onset of OA [ 87 ]. However, these procedures are not well developed, and the long-term maintenance of hyaline cartilage requires further research.

Osteonecrosis of the femoral hip (ONFH) is a refractory disease associated with the collapse of the femoral head and risk of hip arthroplasty in younger populations [ 88 ]. Although total hip arthroplasty (THA) is clinically successful, it is not ideal for young patients, mostly due to the limited lifetime of the prosthesis. An increasing number of clinical studies have evaluated the therapeutic effect of stem cells on ONFH. Most of the authors demonstrated positive outcomes, with reduced pain, improved function, or avoidance of THA [ 89 , 90 , 91 ].

Rejuvenation by cell programming

Ageing is a reversible epigenetic process. The first cell rejuvenation study was published in 2011 [ 92 ]. Cells from aged individuals have different transcriptional signatures, high levels of oxidative stress, dysfunctional mitochondria, and shorter telomeres than in young cells [ 93 ]. There is a hypothesis that when human or mouse adult somatic cells are reprogrammed to iPSCs, their epigenetic age is virtually reset to zero [ 94 ]. This was based on an epigenetic model, which explains that at the time of fertilization, all marks of parenteral ageing are erased from the zygote’s genome and its ageing clock is reset to zero [ 95 ].

In their study, Ocampo et al. [ 96 ] used Oct4, Sox2, Klf4, and C-myc genes (OSKM genes) and affected pancreas and skeletal muscle cells, which have poor regenerative capacity. Their procedure revealed that these genes can also be used for effective regenerative treatment [ 97 ]. The main challenge of their method was the need to employ an approach that does not use transgenic animals and does not require an indefinitely long application. The first clinical approach would be preventive, focused on stopping or slowing the ageing rate. Later, progressive rejuvenation of old individuals can be attempted. In the future, this method may raise some ethical issues, such as overpopulation, leading to lower availability of food and energy.

For now, it is important to learn how to implement cell reprogramming technology in non-transgenic elder animals and humans to erase marks of ageing without removing the epigenetic marks of cell identity.

Cell-based therapies

Stem cells can be induced to become a specific cell type that is required to repair damaged or destroyed tissues (Fig.  6 ). Currently, when the need for transplantable tissues and organs outweighs the possible supply, stem cells appear to be a perfect solution for the problem. The most common conditions that benefit from such therapy are macular degenerations [ 98 ], strokes [ 99 ], osteoarthritis [ 89 , 90 ], neurodegenerative diseases, and diabetes [ 100 ]. Due to this technique, it can become possible to generate healthy heart muscle cells and later transplant them to patients with heart disease.

Stem cell experiments on animals. These experiments are one of the many procedures that proved stem cells to be a crucial factor in future regenerative medicine

In the case of type 1 diabetes, insulin-producing cells in the pancreas are destroyed due to an autoimmunological reaction. As an alternative to transplantation therapy, it can be possible to induce stem cells to differentiate into insulin-producing cells [ 101 ].

Stem cells and tissue banks

iPS cells with their theoretically unlimited propagation and differentiation abilities are attractive for the present and future sciences. They can be stored in a tissue bank to be an essential source of human tissue used for medical examination. The problem with conventional differentiated tissue cells held in the laboratory is that their propagation features diminish after time. This does not occur in iPSCs.

The umbilical cord is known to be rich in mesenchymal stem cells. Due to its cryopreservation immediately after birth, its stem cells can be successfully stored and used in therapies to prevent the future life-threatening diseases of a given patient.

Stem cells of human exfoliated deciduous teeth (SHED) found in exfoliated deciduous teeth has the ability to develop into more types of body tissues than other stem cells [ 102 ] (Table  1 ). Techniques of their collection, isolation, and storage are simple and non-invasive. Among the advantages of banking, SHED cells are:

Guaranteed donor-match autologous transplant that causes no immune reaction and rejection of cells [ 103 ]

Simple and painless for both child and parent

Less than one third of the cost of cord blood storage

Not subject to the same ethical concerns as embryonic stem cells [ 104 ]

In contrast to cord blood stem cells, SHED cells are able to regenerate into solid tissues such as connective, neural, dental, or bone tissue [ 105 , 106 ]

SHED can be useful for close relatives of the donor

Fertility diseases

In 2011, two researchers, Katsuhiko Hayashi et al. [ 107 ], showed in an experiment on mice that it is possible to form sperm from iPSCs. They succeeded in delivering healthy and fertile pups in infertile mice. The experiment was also successful for female mice, where iPSCs formed fully functional eggs .

Young adults at risk of losing their spermatogonial stem cells (SSC), mostly cancer patients, are the main target group that can benefit from testicular tissue cryopreservation and autotransplantation. Effective freezing methods for adult and pre-pubertal testicular tissue are available [ 108 ].

Qiuwan et al. [ 109 ] provided important evidence that human amniotic epithelial cell (hAEC) transplantation could effectively improve ovarian function by inhibiting cell apoptosis and reducing inflammation in injured ovarian tissue of mice, and it could be a promising strategy for the management of premature ovarian failure or insufficiency in female cancer survivors.

For now, reaching successful infertility treatments in humans appears to be only a matter of time, but there are several challenges to overcome. First, the process needs to have high efficiency; second, the chances of forming tumours instead of eggs or sperm must be maximally reduced. The last barrier is how to mature human sperm and eggs in the lab without transplanting them to in vivo conditions, which could cause either a tumour risk or an invasive procedure.

Therapy for incurable neurodegenerative diseases

Thanks to stem cell therapy, it is possible not only to delay the progression of incurable neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease (AD), and Huntington disease, but also, most importantly, to remove the source of the problem. In neuroscience, the discovery of neural stem cells (NSCs) has nullified the previous idea that adult CNS were not capable of neurogenesis [ 110 , 111 ]. Neural stem cells are capable of improving cognitive function in preclinical rodent models of AD [ 112 , 113 , 114 ]. Awe et al. [ 115 ] clinically derived relevant human iPSCs from skin punch biopsies to develop a neural stem cell-based approach for treating AD. Neuronal degeneration in Parkinson’s disease (PD) is focal, and dopaminergic neurons can be efficiently generated from hESCs. PD is an ideal disease for iPSC-based cell therapy [ 116 ]. However, this therapy is still in an experimental phase ( https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4539501 /). Brain tissue from aborted foetuses was used on patients with Parkinson’s disease [ 117 ]. Although the results were not uniform, they showed that therapies with pure stem cells are an important and achievable therapy.

Stem cell use in dentistry

Teeth represent a very challenging material for regenerative medicine. They are difficult to recreate because of their function in aspects such as articulation, mastication, or aesthetics due to their complicated structure. Currently, there is a chance for stem cells to become more widely used than synthetic materials. Teeth have a large advantage of being the most natural and non-invasive source of stem cells.

For now, without the use of stem cells, the most common periodontological treatments are either growth factors, grafts, or surgery. For example, there are stem cells in periodontal ligament [ 118 , 119 ], which are capable of differentiating into osteoblasts or cementoblasts, and their functions were also assessed in neural cells [ 120 ]. Tissue engineering is a successful method for treating periodontal diseases. Stem cells of the root apical areas are able to recreate periodontal ligament. One of the possible methods of tissue engineering in periodontology is gene therapy performed using adenoviruses-containing growth factors [ 121 ].

As a result of animal studies, dentin regeneration is an effective process that results in the formation of dentin bridges [ 122 ].

Enamel is more difficult to regenerate than dentin. After the differentiation of ameloblastoma cells into the enamel, the former is destroyed, and reparation is impossible. Medical studies have succeeded in differentiating bone marrow stem cells into ameloblastoma [ 123 ].

Healthy dental tissue has a high amount of regular stem cells, although this number is reduced when tissue is either traumatized or inflamed [ 124 ]. There are several dental stem cell groups that can be isolated (Fig.  7 ).

Localization of stem cells in dental tissues. Dental pulp stem cells (DPSCs) and human deciduous teeth stem cells (SHED) are located in the dental pulp. Periodontal ligaments stem cells are located in the periodontal ligament. Apical papilla consists of stem cells from the apical papilla (SCAP)

Dental pulp stem cell (DPSC)

These were the first dental stem cells isolated from the human dental pulp, which were [ 125 ] located inside dental pulp (Table  2 ). They have osteogenic and chondrogenic potential. Mesenchymal stem cells (MSCs) of the dental pulp, when isolated, appear highly clonogenic; they can be isolated from adult tissue (e.g. bone marrow, adipose tissue) and foetal (e.g. umbilical cord) [ 126 ] tissue, and they are able to differentiate densely [ 127 ]. MSCs differentiate into odontoblast-like cells and osteoblasts to form dentin and bone. Their best source locations are the third molars [ 125 ]. DPSCs are the most useful dental source of tissue engineering due to their easy surgical accessibility, cryopreservation possibility, increased production of dentin tissues compared to non-dental stem cells, and their anti-inflammatory abilities. These cells have the potential to be a source for maxillofacial and orthopaedic reconstructions or reconstructions even beyond the oral cavity. DPSCs are able to generate all structures of the developed tooth [ 128 ]. In particular, beneficial results in the use of DPSCs may be achieved when combined with other new therapies, such as periodontal tissue photobiomodulation (laser stimulation), which is an efficient technique in the stimulation of proliferation and differentiation into distinct cell types [ 129 ]. DPSCs can be induced to form neural cells to help treat neurological deficits.

Stem cells of human exfoliated deciduous teeth (SHED) have a faster rate of proliferation than DPSCs and differentiate into an even greater number of cells, e.g. other mesenchymal and non-mesenchymal stem cell derivatives, such as neural cells [ 130 ]. These cells possess one major disadvantage: they form a non-complete dentin/pulp-like complex in vivo. SHED do not undergo the same ethical concerns as embryonic stem cells. Both DPSCs and SHED are able to form bone-like tissues in vivo [ 131 ] and can be used for periodontal, dentin, or pulp regeneration. DPSCs and SHED can be used in treating, for example, neural deficits [ 132 ]. DPSCs alone were tested and successfully applied for alveolar bone and mandible reconstruction [ 133 ].

Periodontal ligament stem cells (PDLSCs)

These cells are used in periodontal ligament or cementum tissue regeneration. They can differentiate into mesenchymal cell lineages to produce collagen-forming cells, adipocytes, cementum tissue, Sharpey’s fibres, and osteoblast-like cells in vitro. PDLSCs exist both on the root and alveolar bone surfaces; however, on the latter, these cells have better differentiation abilities than on the former [ 134 ]. PDLSCs have become the first treatment for periodontal regeneration therapy because of their safety and efficiency [ 135 , 136 ].

Stem cells from apical papilla (SCAP)

These cells are mesenchymal structures located within immature roots. They are isolated from human immature permanent apical papilla. SCAP are the source of odontoblasts and cause apexogenesis. These stem cells can be induced in vitro to form odontoblast-like cells, neuron-like cells, or adipocytes. SCAP have a higher capacity of proliferation than DPSCs, which makes them a better choice for tissue regeneration [ 137 , 138 ].

Dental follicle stem cells (DFCs)

These cells are loose connective tissues surrounding the developing tooth germ. DFCs contain cells that can differentiate into cementoblasts, osteoblasts, and periodontal ligament cells [ 139 , 140 ]. Additionally, these cells proliferate after even more than 30 passages [ 141 ]. DFCs are most commonly extracted from the sac of a third molar. When DFCs are combined with a treated dentin matrix, they can form a root-like tissue with a pulp-dentin complex and eventually form tooth roots [ 141 ]. When DFC sheets are induced by Hertwig’s epithelial root sheath cells, they can produce periodontal tissue; thus, DFCs represent a very promising material for tooth regeneration [ 142 ].

Pulp regeneration in endodontics

Dental pulp stem cells can differentiate into odontoblasts. There are few methods that enable the regeneration of the pulp.

The first is an ex vivo method. Proper stem cells are grown on a scaffold before they are implanted into the root channel [ 143 ].

The second is an in vivo method. This method focuses on injecting stem cells into disinfected root channels after the opening of the in vivo apex. Additionally, the use of a scaffold is necessary to prevent the movement of cells towards other tissues. For now, only pulp-like structures have been created successfully.

Methods of placing stem cells into the root channel constitute are either soft scaffolding [ 144 ] or the application of stem cells in apexogenesis or apexification. Immature teeth are the best source [ 145 ]. Nerve and blood vessel network regeneration are extremely vital to keep pulp tissue healthy.

The potential of dental stem cells is mainly regarding the regeneration of damaged dentin and pulp or the repair of any perforations; in the future, it appears to be even possible to generate the whole tooth. Such an immense success would lead to the gradual replacement of implant treatments. Mandibulary and maxillary defects can be one of the most complicated dental problems for stem cells to address.

Acquiring non-dental tissue cells by dental stem cell differentiation

In 2013, it was reported that it is possible to grow teeth from stem cells obtained extra-orally, e.g. from urine [ 146 ]. Pluripotent stem cells derived from human urine were induced and generated tooth-like structures. The physical properties of the structures were similar to natural ones except for hardness [ 127 ]. Nonetheless, it appears to be a very promising technique because it is non-invasive and relatively low-cost, and somatic cells can be used instead of embryonic cells. More importantly, stem cells derived from urine did not form any tumours, and the use of autologous cells reduces the chances of rejection [ 147 ].

Use of graphene in stem cell therapy

Over recent years, graphene and its derivatives have been increasingly used as scaffold materials to mediate stem cell growth and differentiation [ 148 ]. Both graphene and graphene oxide (GO) represent high in-plane stiffness [ 149 ]. Because graphene has carbon and aromatic network, it works either covalently or non-covalently with biomolecules; in addition to its superior mechanical properties, graphene offers versatile chemistry. Graphene exhibits biocompatibility with cells and their proper adhesion. It also tested positively for enhancing the proliferation or differentiation of stem cells [ 148 ]. After positive experiments, graphene revealed great potential as a scaffold and guide for specific lineages of stem cell differentiation [ 150 ]. Graphene has been successfully used in the transplantation of hMSCs and their guided differentiation to specific cells. The acceleration skills of graphene differentiation and division were also investigated. It was discovered that graphene can serve as a platform with increased adhesion for both growth factors and differentiation chemicals. It was also discovered that π-π binding was responsible for increased adhesion and played a crucial role in inducing hMSC differentiation [ 150 ].

Therapeutic potential of extracellular vesicle-based therapies

Extracellular vesicles (EVs) can be released by virtually every cell of an organism, including stem cells [ 151 ], and are involved in intercellular communication through the delivery of their mRNAs, lipids, and proteins. As Oh et al. [ 152 ] prove, stem cells, together with their paracrine factors—exosomes—can become potential therapeutics in the treatment of, e.g. skin ageing. Exosomes are small membrane vesicles secreted by most cells (30–120 nm in diameter) [ 153 ]. When endosomes fuse with the plasma membrane, they become exosomes that have messenger RNAs (mRNAs) and microRNAs (miRNAs), some classes of non-coding RNAs (IncRNAs) and several proteins that originate from the host cell [ 154 ]. IncRNAs can bind to specific loci and create epigenetic regulators, which leads to the formation of epigenetic modifications in recipient cells. Because of this feature, exosomes are believed to be implicated in cell-to-cell communication and the progression of diseases such as cancer [ 155 ]. Recently, many studies have also shown the therapeutic use of exosomes derived from stem cells, e.g. skin damage and renal or lung injuries [ 156 ].

In skin ageing, the most important factor is exposure to UV light, called “photoageing” [ 157 ], which causes extrinsic skin damage, characterized by dryness, roughness, irregular pigmentation, lesions, and skin cancers. In intrinsic skin ageing, on the other hand, the loss of elasticity is a characteristic feature. The skin dermis consists of fibroblasts, which are responsible for the synthesis of crucial skin elements, such as procollagen or elastic fibres. These elements form either basic framework extracellular matrix constituents of the skin dermis or play a major role in tissue elasticity. Fibroblast efficiency and abundance decrease with ageing [ 158 ]. Stem cells can promote the proliferation of dermal fibroblasts by secreting cytokines such as platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), and basic fibroblast growth factor. Huh et al. [ 159 ] mentioned that a medium of human amniotic fluid-derived stem cells (hAFSC) positively affected skin regeneration after longwave UV-induced (UVA, 315–400 nm) photoageing by increasing the proliferation and migration of dermal fibroblasts. It was discovered that, in addition to the induction of fibroblast physiology, hAFSC transplantation also improved diseases in cases of renal pathology, various cancers, or stroke [ 160 , 161 ].

Oh [ 162 ] also presented another option for the treatment of skin wounds, either caused by physical damage or due to diabetic ulcers. Induced pluripotent stem cell-conditioned medium (iPSC-CM) without any animal-derived components induced dermal fibroblast proliferation and migration.

Natural cutaneous wound healing is divided into three steps: haemostasis/inflammation, proliferation, and remodelling. During the crucial step of proliferation, fibroblasts migrate and increase in number, indicating that it is a critical step in skin repair, and factors such as iPSC-CM that impact it can improve the whole cutaneous wound healing process. Paracrine actions performed by iPSCs are also important for this therapeutic effect [ 163 ]. These actions result in the secretion of cytokines such as TGF-β, interleukin (IL)-6, IL-8, monocyte chemotactic protein-1 (MCP-1), vascular endothelial growth factor (VEGF), platelet-derived growth factor-AA (PDGF-AA), and basic fibroblast growth factor (bFGF). Bae et al. [ 164 ] mentioned that TGF-β induced the migration of keratinocytes. It was also demonstrated that iPSC factors can enhance skin wound healing in vivo and in vitro when Zhou et al. [ 165 ] enhanced wound healing, even after carbon dioxide laser resurfacing in an in vivo study.

Peng et al. [ 166 ] investigated the effects of EVs derived from hESCs on in vitro cultured retinal glial, progenitor Müller cells, which are known to differentiate into retinal neurons. EVs appear heterogeneous in size and can be internalized by cultured Müller cells, and their proteins are involved in the induction and maintenance of stem cell pluripotency. These stem cell-derived vesicles were responsible for the neuronal trans-differentiation of cultured Müller cells exposed to them. However, the research article points out that the procedure was accomplished only on in vitro acquired retina.

Challenges concerning stem cell therapy

Although stem cells appear to be an ideal solution for medicine, there are still many obstacles that need to be overcome in the future. One of the first problems is ethical concern.

The most common pluripotent stem cells are ESCs. Therapies concerning their use at the beginning were, and still are, the source of ethical conflicts. The reason behind it started when, in 1998, scientists discovered the possibility of removing ESCs from human embryos. Stem cell therapy appeared to be very effective in treating many, even previously incurable, diseases. The problem was that when scientists isolated ESCs in the lab, the embryo, which had potential for becoming a human, was destroyed (Fig.  8 ). Because of this, scientists, seeing a large potential in this treatment method, focused their efforts on making it possible to isolate stem cells without endangering their source—the embryo.

Use of inner cell mass pluripotent stem cells and their stimulation to differentiate into desired cell types

For now, while hESCs still remain an ethically debatable source of cells, they are potentially powerful tools to be used for therapeutic applications of tissue regeneration. Because of the complexity of stem cell control systems, there is still much to be learned through observations in vitro. For stem cells to become a popular and widely accessible procedure, tumour risk must be assessed. The second problem is to achieve successful immunological tolerance between stem cells and the patient’s body. For now, one of the best ideas is to use the patient’s own cells and devolve them into their pluripotent stage of development.

New cells need to have the ability to fully replace lost or malfunctioning natural cells. Additionally, there is a concern about the possibility of obtaining stem cells without the risk of morbidity or pain for either the patient or the donor. Uncontrolled proliferation and differentiation of cells after implementation must also be assessed before its use in a wide variety of regenerative procedures on living patients [ 167 ].

One of the arguments that limit the use of iPSCs is their infamous role in tumourigenicity. There is a risk that the expression of oncogenes may increase when cells are being reprogrammed. In 2008, a technique was discovered that allowed scientists to remove oncogenes after a cell achieved pluripotency, although it is not efficient yet and takes a longer amount of time. The process of reprogramming may be enhanced by deletion of the tumour suppressor gene p53, but this gene also acts as a key regulator of cancer, which makes it impossible to remove in order to avoid more mutations in the reprogrammed cell. The low efficiency of the process is another problem, which is progressively becoming reduced with each year. At first, the rate of somatic cell reprogramming in Yamanaka’s study was up to 0.1%. The use of transcription factors creates a risk of genomic insertion and further mutation of the target cell genome. For now, the only ethically acceptable operation is an injection of hESCs into mouse embryos in the case of pluripotency evaluation [ 168 ].

Stem cell obstacles in the future

Pioneering scientific and medical advances always have to be carefully policed in order to make sure they are both ethical and safe. Because stem cell therapy already has a large impact on many aspects of life, it should not be treated differently.

Currently, there are several challenges concerning stem cells. First, the most important one is about fully understanding the mechanism by which stem cells function first in animal models. This step cannot be avoided. For the widespread, global acceptance of the procedure, fear of the unknown is the greatest challenge to overcome.

The efficiency of stem cell-directed differentiation must be improved to make stem cells more reliable and trustworthy for a regular patient. The scale of the procedure is another challenge. Future stem cell therapies may be a significant obstacle. Transplanting new, fully functional organs made by stem cell therapy would require the creation of millions of working and biologically accurate cooperating cells. Bringing such complicated procedures into general, widespread regenerative medicine will require interdisciplinary and international collaboration.

The identification and proper isolation of stem cells from a patient’s tissues is another challenge. Immunological rejection is a major barrier to successful stem cell transplantation. With certain types of stem cells and procedures, the immune system may recognize transplanted cells as foreign bodies, triggering an immune reaction resulting in transplant or cell rejection.

One of the ideas that can make stem cells a “failsafe” is about implementing a self-destruct option if they become dangerous. Further development and versatility of stem cells may cause reduction of treatment costs for people suffering from currently incurable diseases. When facing certain organ failure, instead of undergoing extraordinarily expensive drug treatment, the patient would be able to utilize stem cell therapy. The effect of a successful operation would be immediate, and the patient would avoid chronic pharmacological treatment and its inevitable side effects.

Although these challenges facing stem cell science can be overwhelming, the field is making great advances each day. Stem cell therapy is already available for treating several diseases and conditions. Their impact on future medicine appears to be significant.

After several decades of experiments, stem cell therapy is becoming a magnificent game changer for medicine. With each experiment, the capabilities of stem cells are growing, although there are still many obstacles to overcome. Regardless, the influence of stem cells in regenerative medicine and transplantology is immense. Currently, untreatable neurodegenerative diseases have the possibility of becoming treatable with stem cell therapy. Induced pluripotency enables the use of a patient’s own cells. Tissue banks are becoming increasingly popular, as they gather cells that are the source of regenerative medicine in a struggle against present and future diseases. With stem cell therapy and all its regenerative benefits, we are better able to prolong human life than at any time in history.

Abbreviations

Basic fibroblast growth factor

Bone morphogenic proteins

Dental follicle stem cells

Dental pulp stem cells

Embryonic bodies

Embryonic stem cells

Fibroblast growth factors

Good Manufacturing Practice

Graphene oxide

Human amniotic fluid-derived stem cells

Human embryonic stem cells

Human foreskin fibroblasts

Inner cell mass

Non-coding RNA

Induced pluripotent stem cells

In vitro fertilization

Knockout serum replacement

Leukaemia inhibitory factor

Monocyte chemotactic protein-1

Fibroblasts

Messenger RNA

Mesenchymal stem cells of dental pulp

Myogenic differentiation

Osteoarthritis

Octamer-binding transcription factor 3 and 4

Platelet-derived growth factor

Platelet-derived growth factor-AA

Periodontal ligament stem cells

Rho-associated protein kinase

Stem cells from apical papilla

Stem cells of human exfoliated deciduous teeth

Synthetic Serum Substitute

Trophectoderm

Vascular endothelial growth factor

Transforming growth factors

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Wojciech Zakrzewski, Maria Szymonowicz & Zbigniew Rybak

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Zakrzewski, W., Dobrzyński, M., Szymonowicz, M. et al. Stem cells: past, present, and future. Stem Cell Res Ther 10 , 68 (2019). https://doi.org/10.1186/s13287-019-1165-5

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Cloning humans? Biological, ethical, and social considerations

Francisco j ayala.

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Edited by John C. Avise, University of California, Irvine, CA, and approved March 11, 2015 (received for review February 27, 2015)

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Issue date 2015 Jul 21.

There are, in mankind, two kinds of heredity: biological and cultural. Cultural inheritance makes possible for humans what no other organism can accomplish: the cumulative transmission of experience from generation to generation. In turn, cultural inheritance leads to cultural evolution, the prevailing mode of human adaptation. For the last few millennia, humans have been adapting the environments to their genes more often than their genes to the environments. Nevertheless, natural selection persists in modern humans, both as differential mortality and as differential fertility, although its intensity may decrease in the future. More than 2,000 human diseases and abnormalities have a genetic causation. Health care and the increasing feasibility of genetic therapy will, although slowly, augment the future incidence of hereditary ailments. Germ-line gene therapy could halt this increase, but at present, it is not technically feasible. The proposal to enhance the human genetic endowment by genetic cloning of eminent individuals is not warranted. Genomes can be cloned; individuals cannot. In the future, therapeutic cloning will bring enhanced possibilities for organ transplantation, nerve cells and tissue healing, and other health benefits.

Keywords: human origins, natural selection, cultural evolution, genetic therapy, therapeutic cloning

Chimpanzees are the closest relatives of Homo sapiens , our species. There is a precise correspondence bone by bone between the skeletons of a chimpanzee and a human. Humans bear young like apes and other mammals. Humans have organs and limbs similar to birds, reptiles, and amphibians; these similarities reflect the common evolutionary origin of vertebrates. However, it does not take much reflection to notice the distinct uniqueness of our species. Conspicuous anatomical differences between humans and apes include bipedal gait and an enlarged brain. Much more conspicuous than the anatomical differences are the distinct behaviors and institutions. Humans have symbolic language, elaborate social and political institutions, codes of law, literature and art, ethics, and religion; humans build roads and cities, travel by motorcars, ships, and airplanes, and communicate by means of telephones, computers, and televisions.

Human Origins

The hominin lineage diverged from the chimpanzee lineage 6–7 Ma, and it evolved exclusively in the African continent until the emergence of Homo erectus , somewhat before 1.8 Ma. Shortly after its emergence in tropical or subtropical Africa, H. erectus spread to other continents. Fossil remains of H. erectus (sensu lato) are known from Africa, Indonesia (Java), China, the Middle East, and Europe. H. erectus fossils from Java have been dated at 1.81 ± 0.04 and 1.66 ± 0.04 Ma and from Georgia at 1.6–1.8 Ma ( 1 ). Anatomically distinctive H. erectus fossils have been found in Spain, deposited before 780,000 y ago, the oldest in southern Europe ( 2 ).

The transition from H. erectus to H. sapiens occurred around 400,000 y ago, although this date is not well determined owing to uncertainty as to whether some fossils are erectus or archaic forms of sapiens. H. erectus persisted for some time in Asia, until 250,000 y ago in China and perhaps until 100,000 ago in Java, and thus was contemporary with early members of its descendant species, H. sapiens. Fossil remains of Neandertal hominids ( Homo neanderthalensis ), with brains as large as those of H. sapiens , appeared in Europe earlier than 200,000 y ago and persisted until 30,000 or 40,000 y ago ( 3 , 4 ).

There is controversy about the origin of modern humans. Some anthropologists argue that the transition from H. erectus to archaic H. sapiens and later to anatomically modern humans occurred consonantly in various parts of the Old World. Proponents of this “multiregional model” emphasize fossil evidence showing regional continuity in the transition from H. erectus to archaic and then modern H. sapiens . Most anthropologists argue instead that modern humans first arose in Africa somewhat before 100,000 y ago and from there spread throughout the world, eventually replacing elsewhere the preexisting populations of H. erectus , H. neanderthalensis, and archaic H. sapiens . The African origin of modern humans is supported by a wealth of recent genetic evidence and is therefore favored by many evolutionists ( 2 , 4 ).

We know about these matters in three ways: by comparing living primates, including humans, with each other; by discovery and investigation of fossil remains of primates that lived in the past; and by comparing their DNA, proteins, and other molecules. DNA and proteins give us the best information about how closely related we are to each of the primates and those to each other. However, to know how the human lineage changed in anatomy and behavior over time as our ancestors became more and more human-like, we have to study fossils and the tools they used and made, as well as other remnants of their activities ( 2 , 5 ).

Humans live in groups that are socially organized and so do other primates. However, other primate societies do not approach the complexity of human social organization. A distinctive human social trait is culture, which may be understood as the set of nonstrictly biological human activities and creations. Culture includes social and political institutions, ways of doing things, religious and ethical traditions, language, common sense and scientific knowledge, art and literature, technology, and in general all of the creations of the human mind. The advent of culture has brought with it cultural evolution, a superorganic mode of evolution superimposed on the organic mode, that has become the dominant mode of human evolution. Cultural evolution has come about because of cultural inheritance, a distinctively human mode of achieving adaptation to the environment ( 2 , 6 , 7 ).

There are in mankind two kinds of heredity: the biological and the cultural. Biological inheritance in humans is very much like that in any other sexually reproducing organism; it is based on the transmission of genetic information encoded in DNA from one generation to the next by means of the sex cells. Cultural inheritance, on the other hand, is based on transmission of information by a teaching-learning process, which is in principle independent of biological parentage. Culture is transmitted by instruction and learning, by example and imitation, through books, newspapers, radio, television, and motion pictures, through works of art, and through any other means of communication. Culture is acquired by every person from parents, relatives, and neighbors and from the whole human environment. Acquired cultural traits may be beneficial but also toxic; for example, racial prejudice or religious bigotry.

Biological heredity is Mendelian or vertical; it is transmitted from parents to their children, and only inherited traits can be transmitted to the progeny. (New mutations are insignificant in the present context.) Cultural heredity is Lamarckian: acquired characters can be transmitted to the progeny. However, cultural heredity goes beyond Lamarckian heredity, because it is horizontal and oblique and not only vertical. Traits can be acquired from and transmitted to other members of the same generation, whether or not they are relatives, and also from and to all other individuals with whom a person has contact, whether they are from the same or from any previous or ensuing generation.

Cultural inheritance makes possible for people what no other organism can accomplish—the cumulative transmission of experience from generation to generation. Animals can learn from experience, but they do not transmit their experiences or their discoveries (at least not to any large extent) to the following generations. Animals have individual memory, but they do not have a “social memory.” Humans, on the other hand, have developed a culture because they can transmit cumulatively their experiences from generation to generation.

Cultural inheritance makes possible cultural evolution, a new mode of adaptation to the environment that is not available to nonhuman organisms. Organisms in general adapt to the environment by means of natural selection, by changing over generations their genetic constitution to suit the demands of the environment. However, humans, and humans alone, can also adapt by changing the environment to suit the needs of their genes. (Animals build nests and modify their environment also in other ways, but the manipulation of the environment by any nonhuman species is trivial compared with mankind's manipulation.) For the last few millennia, humans have been adapting the environments to their genes more often than their genes to the environments.

To extend its geographical habitat, or to survive in a changing environment, a population of organisms must become adapted, through slow accumulation of genetic variants sorted out by natural selection, to the new climatic conditions, different sources of food, different competitors, and so on. The discovery of fire and the use of shelter and clothing allowed humans to spread from the warm tropical and subtropical regions of the Old World to the whole Earth, except for the frozen wastes of Antarctica, without the anatomical development of fur or hair. Humans did not wait for genetic mutants promoting wing development; they have conquered the air in a somewhat more efficient and versatile way by building flying machines. People travel the rivers and the seas without gills or fins. The exploration of outer space has started without waiting for mutations providing humans with the ability to breathe with low oxygen pressures or to function in the absence of gravity; astronauts carry their own oxygen and specially equipped pressure suits. From their obscure beginnings in Africa, humans have become the most widespread and abundant species of mammal on earth. It was the appearance of culture as a superorganic form of adaptation that made mankind the most successful animal species.

Cultural adaptation has prevailed in mankind over biological adaptation because it is a more effective mode of adaptation; it is more rapid and it can be directed. A favorable genetic mutation newly arisen in an individual can be transmitted to a sizeable part of the human species only through innumerable generations. However, a new scientific discovery or technical achievement can be transmitted to the whole of mankind, potentially at least, in less than one generation. Witness the rapid spread of personal computers, iPhones, and the Internet. Moreover, whenever a need arises, culture can directly pursue the appropriate changes to meet the challenge. On the contrary, biological adaptation depends on the accidental availability of a favorable mutation, or of a combination of several mutations, at the time and place where the need arises ( 2 , 6 , 7 ).

Biological Evolution in Modern Humans

There is no scientific basis to the claim sometimes made that the biological evolution of mankind has stopped, or nearly so, at least in technologically advanced countries. It is asserted that the progress of medicine, hygiene, and nutrition have largely eliminated death before middle age; that is, most people live beyond reproductive age, after which death is inconsequential for natural selection. That mankind continues to evolve biologically can be shown because the necessary and sufficient conditions for biological evolution persist. These conditions are genetic variability and differential reproduction. There is a wealth of genetic variation in mankind. With the trivial exception of identical twins, developed from a single fertilized egg, no two people who live now, lived in the past, or will live in the future, are likely to be genetically identical. Much of this variation is relevant to natural selection ( 5 , 8 , 9 ).

Natural selection is simply differential reproduction of alternative genetic variants. Natural selection will occur in mankind if the carriers of some genotypes are likely to leave more descendants than the carriers of other genotypes. Natural selection consists of two main components: differential mortality and differential fertility; both persist in modern mankind, although the intensity of selection due to postnatal mortality has been somewhat attenuated.

Death may occur between conception and birth (prenatal) or after birth (postnatal). The proportion of prenatal deaths is not well known. Death during the early weeks of embryonic development may go totally undetected. However, it is known that no less than 20% of all ascertained human conceptions end in spontaneous abortion during the first 2 mo of pregnancy. Such deaths are often due to deleterious genetic constitutions, and thus they have a selective effect in the population. The intensity of this form of selection has not changed substantially in modern mankind, although it has been slightly reduced with respect to a few genes such as those involved in Rh blood group incompatibility.

Postnatal mortality has been considerably reduced in recent times in technologically advanced countries. For example, in the United States, somewhat less than 50% of those born in 1840 survived to age 45, whereas the average life expectancy for people born in the United States in 1960 is 78 y ( Table 1 ) ( 8 , 10 ). In some regions of the world, postnatal mortality remains quite high, although there it has also generally decreased in recent decades. Mortality before the end of reproductive age, particularly where it has been considerably reduced, is largely associated with genetic defects, and thus it has a favorable selective effect in human populations. Several thousand genetic variants are known that cause diseases and malformations in humans; such variants are kept at low frequencies due to natural selection.

Percent of Americans born between 1840 and 1960 surviving to ages 15 and 45

Reprinted from ref. 8 .

It might seem at first that selection due to differential fertility has been considerably reduced in industrial countries as a consequence of the reduction in the average number of children per family that has taken place. However, this is not so. The intensity of fertility selection depends not on the mean number of children per family, but on the variance in the number of children per family. It is clear why this should be so. Assume that all people of reproductive age marry and that all have exactly the same number of children. In this case, there would not be fertility selection whether couples all had very few or all had very many children. Assume, on the other hand, that the mean number of children per family is low, but some families have no children at all or very few, whereas others have many. In this case, there would be considerable opportunity for selection—the genotypes of parents producing many children would increase in frequency at the expense of those having few or none. Studies of human populations have shown that the opportunity for natural selection often increases as the mean number of children decreases. An extensive study published years ago showed that the index of opportunity for selection due to fertility was four times larger among United States women born in the 20th century, with an average of less than three children per woman, than among women in the Gold Coast of Africa or in rural Quebec, who had three times or more children on average ( Table 2 ) ( 8 , 11 ). There is no evidence that natural selection due to fertility has decreased in modern human populations.

Mean number of children per family and index of opportunity for fertility selection I f , in various human populations

I f is calculated as the variance divided by the square of the mean number of children. The opportunity for selection usually increases as the mean number of children decreases. Reprinted from ref. 8 .

Natural selection may decrease in intensity in the future, but it will not disappear altogether. As long as there is genetic variation and the carriers of some genotypes are more likely to reproduce than others, natural selection will continue operating in human populations. Cultural changes, such as the development of agriculture, migration from the country to the cities, environmental pollution, and many others, create new selective pressures. The pressures of city life are partly responsible for the high incidence of mental disorders in certain human societies. The point to bear in mind is that human environments are changing faster than ever owing precisely to the accelerating rate of cultural change, and environmental changes create new selective pressures, thus fueling biological evolution.

Natural selection is the process of differential reproduction of alternative genetic variants. In terms of single genes, variation occurs when two or more alleles are present in the population at a given gene locus. How much genetic variation exists in the current human population? The answer is “quite a lot,” as will be presently shown, but natural selection will take place only if the alleles of a particular gene have different effects on fitness; that is, if alternative alleles differentially impact the probability of survival and reproduction.

The two genomes that we inherit from each parent are estimated to differ at about one or two nucleotides per thousand. The human genome consists of somewhat more than 3 billion nucleotides ( 12 ). Thus, about 3–6 million nucleotides are different between the two genomes of each human individual, which is a lot of genetic polymorphism. Moreover, the process of mutation introduces new variation in any population every generation. The rate of mutation in the human genome is estimated to be about 10 −8 , which is one nucleotide mutation for every hundred million nucleotides, or about 30 new mutations per genome per generation. Thus, every human has about 60 new mutations (30 in each genome) that were not present in the parents. If we consider the total human population, that is 60 mutations per person multiplied by 7 billion people, which is about 420 billion new mutations per generation that are added to the preexisting 3–6 million polymorphic nucleotides per individual.

That is a lot of mutations, even if many are redundant. Moreover, we must remember that the polymorphisms that count for natural selection are those that impact the probability of survival and reproduction of their carriers. Otherwise, the variant nucleotides may increase or decrease in frequency by chance, a process that evolutionists call “genetic drift,” but will not be impacted by natural selection ( 2 , 12 , 13 ).

Genetic Disorders

More than 2,000 human diseases and abnormalities that have a genetic causation have been identified in the human population. Genetic disorders may be dominant, recessive, multifactorial, or chromosomal. Dominant disorders are caused by the presence of a single copy of the defective allele, so that the disorder is expressed in heterozygous individuals: those having one normal and one defective allele. In recessive disorders, the defective allele must be present in both alleles, that is, it is inherited from each parent to be expressed. Multifactorial disorders are caused by interaction among several gene loci; chromosomal disorders are due to the presence or absence of a full chromosome or a fragment of a chromosome ( 14 , 15 ).

Examples of dominant disorders are some forms of retinoblastoma and other kinds of blindness, achondroplastic dwarfism, and Marfan syndrome (which is thought to have affected President Lincoln). Examples of recessive disorders are cystic fibrosis, Tay-Sachs disease, and sickle cell anemia (caused by an allele that in heterozygous condition protects against malaria). Examples of multifactorial diseases are spina bifida and cleft palate. Among the most common chromosomal disorders are Down syndrome, caused by the presence of an extra chromosome 21, and various kinds due to the absence of one sex chromosome or the presence of an extra one, beyond the normal condition of XX for women and XY for men. Examples are Turner’s syndrome (XO) and Klinefelter’s syndrome (XXY) ( 16 ).

The incidence of genetic disorders expressed in the living human population is estimated to be no less than 2.56%, impacting about 180 million people. Natural selection reduces the incidence of the genes causing disease, more effectively in the case of dominant disorders, where all carriers of the gene will express the disease, than for recessive disorders, which are expressed only in homozygous individuals. Consider, for example, phenylketonuria (PKU), a lethal disease if untreated, due to homozygosis for a recessive gene, which has an incidence of 1 in 10,000 newborns or 0.01%. PKU is due to an inability to metabolize the amino acid phenylalanine with devastating mental and physical effects. A very elaborate diet free of phenylalanine allows the patient to survive and reproduce if started early in life. The frequency of the PKU allele is about 1%, so that in heterozygous conditions it is present in more than 100 million people, but only the 0.01% of people who are homozygous express the disease and are subject to natural selection. The reduction of genetic disorders due to natural selection is balanced with their increase due to the incidence of new mutations.

Let’s consider another example. Hereditary retinoblastoma is a disease attributed to a dominant mutation of the gene coding for the retinoblastoma protein, RB1, but it is actually due to a deletion in chromosome 13. The unfortunate child with this condition develops a tumorous growth during infancy that, without treatment, starts in one eye and often extends to the other eye and then to the brain, causing death before puberty. Surgical treatment now makes it possible to save the life of the child if the condition is detected sufficiently early, although often one or both eyes may be lost. The treated person can live a more or less normal life, marry, and procreate. However, because the genetic determination is dominant (a gene deletion), one half of the progeny will, on the average, be born with the same genetic condition and will have to be treated. Before modern medicine, every mutation for retinoblastoma arising in the human population was eliminated from the population in the same generation owing to the death of its carrier. With surgical treatment, the mutant condition can be preserved, and new mutations arising each generation are added to those arisen in the past (refs. 17 and 18 ; www.abedia.com/wiley/index.html ).

The proportion of individuals affected by any one serious hereditary infirmity is relatively small, but there are more than 2,000 known serious physical infirmities determined by genes. When all these hereditary ailments are considered together, the proportion of persons born who will suffer from a serious handicap during their lifetimes owing to their heredity is more than 2% of the total population, as pointed out above (refs. 15 , 16 , and 19 ; www.abedia.com/wiley/index.html ).

The problem becomes more serious when mental defects are taken into consideration. More than 2% of the population is affected by schizophrenia or a related condition known as schizoid disease, ailments that may be in some cases determined by a single mutant gene. Another 3% or so of the population suffer from mild mental retardation (IQ less than 70). More than 100 million people in the world suffer from mental impairments due in good part to the genetic endowment they inherited from their parents.

Natural selection also acts on a multitude of genes that do not cause disease. Genes impact skin pigmentation, hair color and configuration, height, muscle strength and body shape, and many other anatomical polymorphisms that are apparent, as well as many that are not externally obvious, such as variations in the blood groups, in the immune system, and in the heart, liver, kidney, pancreas, and other organs. It is not always known how natural selection impacts these traits, but surely it does and does it differently in different parts of the world or at different times, as a consequence of the development of new vaccines, drugs, and medical treatments, and also as a consequence of changes in lifestyle, such as the reduction of the number of smokers or the increase in the rate of obesity in a particular country.

Genetic Therapy

Where is human evolution going? Biological evolution is directed by natural selection, which is not a benevolent force guiding evolution toward sure success. Natural selection brings about genetic changes that often appear purposeful because they are dictated by the requirements of the environment. The end result may, nevertheless, be extinction—more than 99.9% of all species that ever existed have become extinct. Natural selection has no purpose; humans alone have purposes and they alone may introduce them into their evolution. No species before mankind could select its evolutionary destiny; mankind possesses techniques to do so, and more powerful techniques for directed genetic change are becoming available. Because we are self-aware, we cannot refrain from asking what lies ahead, and because we are ethical beings, we must choose between alternative courses of action, some of which may appear as good and others as bad.

The argument has been advanced that the biological endowment of mankind is rapidly deteriorating owing precisely to the improving conditions of life and to the increasing power of modern medicine. The detailed arguments that support this contention involve some mathematical exercises, but their essence can be simply presented. Genetic changes (i.e., point or chromosome mutations) arise spontaneously in humans and in other living species. The great majority of newly arising mutations are either neutral or harmful to their carriers; only a very small fraction are likely to be beneficial. In a human population under the so-called “natural” conditions, that is, without the intervention of modern medicine and technology, the newly arising harmful mutations are eliminated from the population more or less rapidly depending on how harmful they are. The more harmful the effect of a mutation, the more rapidly it will be eliminated from the population by the process of natural selection. However, owing to medical intervention and, more recently, because of the possibility of genetic therapy, the elimination of some harmful mutations from the population is no longer taking place as rapidly and effectively as it did in the past.

Molecular biology has introduced in modern medicine a new way to cure diseases, namely genetic therapy, direct intervention in the genetic makeup of an individual. Gene therapy can be somatic or germ line. Germ-line genetic therapy would seek to correct a genetic defect, not only in the organs or tissues impacted, but also in the germ line, so that the person treated would not transmit the genetic impairment to the descendants. As of now, no interventions of germ-line therapy are seriously sought by scientists, physicians, or pharmaceutical companies.

The possibility of gene therapy was first anticipated in 1972 ( 20 ). The possible objectives are to correct the DNA of a defective gene or to insert a new gene that would allow the proper function of the gene or DNA to take place. In the case of a harmful gene, the objective would be to disrupt the gene that is not functioning properly.

The eminent biologist E. O. Wilson (2014) has stated, many would think somewhat hyperbolically, that the issue of how much to use genetic engineering to direct our own evolution, is “the greatest moral dilemma since God stayed the hand of Abraham” ( 21 ).

The first successful interventions of gene therapy concerned patients suffering from severe combined immunodeficiency (SCID), first performed in a 4-y-old girl at the National Institutes of Health in 1990 ( 22 ), soon followed by successful trials in other countries ( 23 ). Treatments were halted temporarily from 2000 to 2002 in Paris, when 2 of about 12 treated children developed a leukemia-like condition, which was indeed attributed to the gene therapy treatment. Since 2004, successful clinical trials for SCID have been performed in the United States, United Kingdom, France, Italy, and Germany ( 24 , 25 ).

Gene therapy treatments are still considered experimental. Successful clinical trials have been performed in patients suffering from adrenoleukodystrophy, Parkinson’s disease, chronic lymphocytic leukemia, acute lymphocytic leukemia, multiple myeloma, and hemophilia ( 26 , 27 ). Initially, the prevailing gene therapy methods involved recombinant viruses, but nonviral methods (transfection molecules) have become increasingly successful. Since 2013, US pharmaceutical companies have invested more than $600 million in gene therapy ( 28 ). However, in addition to the huge economic costs, technical hurdles remain. Frequent negative effects include immune response against an extraneous object introduced into human tissues, leukemia, tumors, and other disorders provoked by vector viruses. Moreover, the genetic therapy corrections are often short lived, which calls for multiple rounds of treatment, thereby increasing costs and other handicaps. In addition, many of the most common genetic disorders are multifactorial and are thus beyond current gene therapy treatment. Examples are diabetes, high blood pressure, heart disease, arthritis, and Alzheimer’s disease, which at the present state of knowledge and technology are not suitable for gene therapy.

If a genetic defect is corrected in the affected cells, tissues, or organs, but not in the germ line, the ova or sperm produced by the individual will transmit the defect to the progeny. A deleterious gene that might have been reduced in frequency or eliminated from the population, owing to the death or reduced fertility of the carrier, will now persist in the population and be added to its load of hereditary diseases. A consequence of genetic therapy is that the more hereditary diseases and defects are cured today, the more of them will be there to be cured in the succeeding generations. This consequence follows not only from gene therapy but also from typical medical treatments.

The Nobel laureate geneticist H. J. Muller eloquently voiced this concern about the cure, whether through genetic therapy or traditional medical treatment, of genetic ailments. “The more sick people we now cure and allow them to reproduce, the more there will be to cure in the future.” The fate toward which mankind is drifting is painted by Muller in somber colors. “The amount of genetically caused impairment suffered by the average individual…must by that time have grown….[P]eople’s time and energy…would be devoted chiefly to the effort to live carefully, to spare and to prop up their own feebleness, to soothe their inner disharmonies and, in general, to doctor themselves as effectively as possible. For everyone would be an invalid, with his own special familial twists….” (ref. 29 ; Fig. 1 ).

The bionic human, on the cover of Science : an image that could represent how H. J. Muller anticipates the human condition, a few centuries hence, showing the accumulation of physical handicaps as a consequence of the medical cure of hereditary diseases. Image by Cameron Slayden and Nathalie Cary; reprinted with permission from AAAS.

It must be pointed out that the population genetic consequences of curing hereditary diseases are not as immediate (“a few centuries hence”) as Muller anticipates. Consider, as a first example, we look at the recessive hereditary condition of PKU. The estimated frequency of the gene is q = 0.01; the expected number of humans born with PKU is q 2 = 0.0001, 1 for every 10,000 births. If all PKU individuals are cured all over the world and all of them leave as many descendants, on the average, as other humans, the frequency of the PKU allele will double after 1/q = 1/0.01 = 100 generations. If we assume 25 y per generation, we conclude that after 2,500 y, the frequency of the PKU allele will be q = 0.02, and q 2 = 0.0004, so that 4 of every 10,000 persons, rather than only 1, will be born with PKU.

In the case of dominant lethal diseases, the incidence is determined by the mutation frequency of the normal to the disease allele, which is typically of the order of m = 10 −6 –10 −8 , or between one in a million and one in one hundred million. Assuming the highest rate of m = 10 −6 , the incidence of the disease after 100 generations will become 1 for every 10,000 births. It would therefore seem likely that much earlier than 2,500 y, humans are likely to find ways of correcting hereditary ailments in the germ line, thereby stopping their transmission.

It must be pointed out that, although the proportion of individuals affected by any one serious hereditary infirmity is relatively small, there are many such hereditary ailments, which on the aggregate make the problem very serious. The problem becomes more serious when mental defects are taken into consideration. As pointed out above, more than 100 million people in the world suffer from mental impairments due in good part to the genetic endowment they inherited from their parents.

Human cloning may refer to “therapeutic cloning,” particularly the cloning of embryonic cells to obtain organs for transplantation or for treating injured nerve cells and other health purposes. Human cloning more typically refers to “reproductive cloning,” the use of somatic cell nuclear transfer (SCNT) to obtain eggs that could develop into adult individuals.

Human cloning has occasionally been suggested as a way to improve the genetic endowment of mankind, by cloning individuals of great achievement, for example, in sports, music, the arts, science, literature, politics, and the like, or of acknowledged virtue. These suggestions seemingly have never been taken seriously. However, some individuals have expressed a wish, however unrealistic, to be cloned, and some physicians have on occasion advertised that they were ready to carry out the cloning ( 30 ). The obstacles and drawbacks are many and insuperable, at least at the present state of knowledge.

Biologists use the term cloning with variable meanings, although all uses imply obtaining copies more or less precise of a biological entity. Three common uses refer to cloning genes, cloning cells, and cloning individuals. Cloning an individual, particularly in the case of a multicellular organism, such as a plant or an animal, is not strictly possible. The genes of an individual, the genome, can be cloned, but the individual itself cannot be cloned, as it will be made clear below.

Cloning genes or, more generally, cloning DNA segments is routinely done in many genetics and pharmaceutical laboratories throughout the world ( 12 , 31 ). Technologies for cloning cells in the laboratory are seven decades old and are used for reproducing a particular type of cell, for example a skin or a liver cell, in order to investigate its characteristics.

Individual human cloning occurs naturally in the case of identical twins, when two individuals develop from a single fertilized egg. These twins are called identical, precisely because they are genetically identical to each other.

The sheep Dolly, cloned in July 1996, was the first mammal artificially cloned using an adult cell as the source of the genotype. Frogs and other amphibians were obtained by artificial cloning as early as 50 y earlier ( 32 ).

Cloning an animal by SCNT proceeds as follows. First, the genetic information in the egg of a female is removed or neutralized. Somatic (i.e., body) cells are taken from the individual selected to be cloned, and the cell nucleus (where the genetic information is stored) of one cell is transferred with a micropipette into the host oocyte. The egg, so “fertilized,” is stimulated to start embryonic development ( 33 ).

Can a human individual be cloned? The correct answer is, strictly speaking, no. What is cloned are the genes, not the individual; the genotype, not the phenotype. The technical obstacles are immense even for cloning a human’s genotype.

Ian Wilmut, the British scientist who directed the cloning project, succeeded with Dolly only after 270 trials. The rate of success for cloning mammals has notably increased over the years without ever reaching 100%. The animals presently cloned include mice, rats, goats, sheep, cows, pigs, horses, and other mammals. The great majority of pregnancies end in spontaneous abortion ( 34 ). Moreover, as Wilmut noted, in many cases, the death of the fetus occurs close to term, with devastating economic, health, and emotional consequences in the case of humans ( 35 ).

In mammals, in general, the animals produced by cloning suffer from serious health handicaps, among others, gross obesity, early death, distorted limbs, and dysfunctional immune systems and organs, including liver and kidneys, and other mishaps. Even Dolly had to be euthanized early in 2003, after only 6 y of life, because her health was rapidly decaying, including progressive lung disease and arthritis ( 35 , 36 ).

The low rate of cloning success may improve in the future. It may be that the organ and other failures of those that reach birth will be corrected by technical advances. Human cloning would still face ethical objections from a majority of concerned people, as well as opposition from diverse religions. Moreover, there remains the limiting consideration asserted earlier: it might be possible to clone a person’s genes, but the individual cannot be cloned. The character, personality, and the features other than anatomical and physiological that make up the individual are not precisely determined by the genotype.

The Genotype and the Individual

The genetic makeup of an individual is its genotype. The phenotype refers to what the individual is, which includes not only the individual’s external appearance or anatomy, but also its physiology, as well as behavioral predispositions and attributes, encompassing intellectual abilities, moral values, aesthetic preferences, religious values, and, in general, all other behavioral characteristics or features, acquired by experience, imitation, learning, or in any other way throughout the individual’s life, from conception to death. The phenotype results from complex networks of interactions between the genes and the environment.

A person’s environmental influences begin, importantly, in the mother’s womb and continue after birth, through childhood, adolescence, and the whole life. Impacting behavioral experiences are associated with family, friends, schooling, social and political life, readings, aesthetic and religious experiences, and every event in the person’s life, whether conscious or not. The genotype of a person has an unlimited number, virtually infinite, of possibilities to be realized, which has been called the genotype’s “norm of reaction,” only one of which will be the case in a particular individual ( 37 ). If an adult person is cloned, the disparate life circumstances experienced many years later would surely result in a very different individual, even if anatomically the individual would resemble the genome’s donor at a similar age.

An illustration of environmental effects on the phenotype, and of interactions between the genotype and the environment, is shown in Fig. 2 ( 38 ). Three plants of the cinquefoil, Potentilla glandulosa , were collected in California—one on the coast at about 100 ft above sea level (Stanford), the second at about 4,600 ft (Mather), and the third in the Alpine zone of the Sierra Nevada at about 10,000 ft above sea level (Timberline). From each plant, three cuttings were obtained in each of several replicated experiments, which were planted in three experimental gardens at different altitudes, the same gardens from which the plants were collected. The division of one plant ensured that all three cuttings planted at different altitudes had the same genotype; that is, they were genetic clones from one another. ( P. glandulosa , like many other plants, can be reproduced by cuttings, which are genetically identical.)

Interacting effects of the genotype and the environment on the phenotype of the cinquefoil Pontentilla glandulosa . Cuttings of plants collected at different altitudes were planted in three different experimental gardens. Plants in the same row are genetically identical because they have been grown from cuttings of a single plant; plants in the same column are genetically different but have been grown in the same experimental garden. Reprinted with permission from ref. 13 .

Comparison of the plants in any row shows how a given genotype gives rise to different phenotypes in different environments. Genetically identical plants (for example, those in the bottom row) may prosper or not, even die, depending on the environmental conditions. Plants from different altitudes are known to be genetically different. Hence, comparison of the plants in any column shows that in a given environment, different genotypes result in different phenotypes. An important inference derived from this experiment is that there is no single genotype that is best in all environments.

The interaction between the genotype and the environment is similarly significant, or even more so, in the case of animals. In one experiment, two strains of rats were selected over many generations; one strain for brightness at finding their way through a maze and the other for dullness ( Fig. 3 ; ref. 39 ). Selection was done in the bright strain by using the brightest rats of each generation to breed the following generation, and in the dull strain by breeding the dullest rats of every generation. After many generations of selection, the descendant bright rats made only about 120 errors running through the maze, whereas dull rats averaged 165 errors. That is a 40% difference. However, the differences between the strains disappeared when rats of both strains were raised in an unfavorable environment of severe deprivation, where both strains averaged 170 errors. The differences also nearly disappeared when the rats were raised with abundant food and other favorable conditions. In this optimal environment, the dull rats reduced their average number of errors from 165 to 120. As with the cinquefoil plants, we see ( i ) that a given genotype gives rise to different phenotypes in different environments and ( ii ) that the differences in phenotype between two genotypes change from one environment to another—the genotype that is best in one environment may not be best in another.

Results of an experiment with two strains of rats: one selected for brightness and the other for dullness. After many generations of selection, when raised in the same environment in which the selection was practiced (normal), bright rats made about 45 fewer errors than dull rats in the maze used for the tests. However, when the rats were raised in an impoverished (restricted) environment, bright and dull rats made the same number of errors. When raised in an abundant (stimulating) environment, the two strains performed nearly equally well. Reprinted with permission from ref. 13 .

Cloning Humans?

In the second half of the 20th century, as dramatic advances were taking place in genetic knowledge, as well as in the genetic technology often referred to as “genetic engineering,” some utopian proposals were advanced, at least as suggestions that should be explored and considered as possibilities, once the technologies had sufficiently progressed. Some proposals suggested that persons of great intellectual or artistic achievement or of great virtue be cloned. If this was accomplished in large numbers, the genetic constitution of mankind would, it was argued, considerably improve.

Such utopian proposals are grossly misguided. It should be apparent that, as stated above, it is not possible to clone a human individual. Seeking to multiply great benefactors of humankind, such as persons of great intelligence or character, we might obtain the likes of Stalin, Hitler, or Bin Laden. As the Nobel Laureate geneticist George W. Beadle asserted many years ago: “Few of us would have advocated preferential multiplication of Hitler’s genes. Yet who can say that in a different cultural context Hitler might not have been one of the truly great leaders of men, or that Einstein might not have been a political villain” ( 8 ). There is no reason whatsoever to expect that the genomes of individuals with excellent attributes would, when cloned, produce individuals similarly endowed with virtue or intelligence. Identical genomes yield, in different environments, individuals who may be quite different. Environments cannot be reproduced, particularly several decades apart, which would be the case when the genotype of the persons selected because of their eminent achievement might be cloned.

Are there circumstances that would justify cloning a person, because he or she wants it? One might think of a couple unable to have children, or a man or woman who does not want to marry, or of two lesbian lovers who want to have a child with the genotype of one in an ovum of the other, or of other special cases that might come to mind ( 40 ). It must be, first, pointed out that the cloning technology has not yet been developed to an extent that would make possible to produce a healthy human individual by cloning. Second, and most important, the individual produced by cloning would be a very different person from the one whose genotype is cloned, as belabored above.

Ethical, social, and religious values will come into play when seeking to decide whether a person might be allowed to be cloned. Most people are likely to disapprove. Indeed, many countries have prohibited human cloning. In 2004, the issue of cloning was raised in several countries where legislatures were also considering whether research on embryonic stem cells should be supported or allowed. The Canadian Parliament on March 12, 2004 passed legislation permitting research with stem cells from embryos under specific conditions, but human cloning was banned, and the sale of sperm and payments to egg donors and surrogate mothers were prohibited. The French Parliament on July 9, 2004 adopted a new bioethics law that allows embryonic stem cell research but considers human cloning a “crime against the human species.” Reproductive cloning experiments would be punishable by up to 20 y in prison. Japan’s Cabinet Council for Science and Technology Policy voted on July 23, 2004 to adopt policy recommendations that would permit the limited cloning of human embryos for scientific research but not the cloning of individuals. On January 14, 2001, the British government amended the Human Fertilization and Embryology Act of 1990 by allowing embryo research on stem cells and allowing therapeutic cloning. The Human Fertilization and Embryology Act of 2008 explicitly prohibited reproductive cloning but allowed experimental stem cell research for treating diabetes, Parkinson’s disease, and Alzheimer’s disease ( 41 , 42 ). On February 3, 2014, the House of Commons voted to legalize a gene therapy technique known as mitochondrial replacement, or three-person in vitro fertilization, in which mitochondria from a donor’s egg cell contribute to a couple’s embryo ( 43 ). In the United States, there are currently no federal laws that ban cloning completely ( 42 ). Thirteen states (Arkansas, California, Connecticut, Iowa, Indiana, Massachusetts, Maryland, Michigan, North Dakota, New Jersey, Rhode Island, South Dakota, and Virginia) ban reproductive cloning, and three states (Arizona, Maryland, and Missouri) prohibit use of public funds for research on reproductive cloning ( 44 ).

Therapeutic Cloning

Cloning of embryonic cells (stem cells) could have important health applications in organ transplantation, treating injured nerve cells, and otherwise. In addition to SCNT, the method discussed above for cloning individuals, another technique is available, induced pluripotent stem cells (iPSCs), although SCNT has proven to be much more effective and less costly. The objective is to obtain pluripotent stem cells that have the potential to differentiate in any of the three germ layers characteristic of humans and other animals: endoderm (lungs and interior lining of stomach and gastrointestinal tract), ectoderm (nervous systems and epidermal tissues), and mesoderm (muscle, blood, bone, and urogenital tissues). Stem cells, with more limited possibilities than pluripotent cells, can also be used for specific therapeutic purposes ( 45 ).

Stem cell therapy consists of cloning embryonic cells to obtain pluripotent or other stem cells that can be used in regenerative medicine, to treat or prevent all sorts of diseases, and for the transplantation of organs. At present, bone marrow transplantation is a widely used form of stem cell therapy; stem blood cells are used in the treatment of sickle cell anemia, a lethal disease when untreated, which is very common in places where malaria is rife because heterozygous individuals are protected against infection by Plasmodium falciparum , the agent of malignant malaria. One of the most promising applications of therapeutic cloning is the growth of organs for transplantation, using stem cells that have the genome of the organ recipient. Two major hurdles would be overcome. One is the possibility of immune rejection; the other is the availability of organs from suitable donors. Another regenerative medical application that might be anticipated is the therapeutic growth of nerve cells. There are hundreds of thousands of individuals throughout the world paralyzed from the neck down and confined for life to a wheelchair as a consequence of damage to the spinal cord below the neck, often as a consequence of a car accident or a fall, that interrupts the transmission of nerve activity from the brain to the rest of the body and vice versa. A small growth of nerve cells sufficient to heal the wound in the spinal cord would have enormous health consequences for the wounded persons and for society.

At present, the one gene therapy modification of the embryo that can be practiced is mitochondrial replacement (MR), legalized in the United Kingdom by the House of Commons on February 3, 2014 ( 43 ), as mentioned earlier. Mutations in the mitochondrial DNA of about 1 in 6,500 individuals account for a variety of severe and often fatal conditions, including blindness, muscular weakness, and heart failure ( 46 ). With MR, the embryo possesses nuclear DNA from the mother and father, as well as mtDNA from a donor female who has healthy mtDNA. However, MR remains technically challenging, with a low rate of success. One complicating issue is that mtDNA replacement is not 100% successful; disease-causing mutant mtDNA persists in the developing embryo and may account for eventual diseases due to heteroplasmy, at least in some tissues. A second issue of concern is that mtDNA disorders often appear late in life. It remains unknown whether the benefits of MR as currently practiced may persist in advanced age.

The author declares no conflict of interest.

This paper results from the Arthur M. Sackler Colloquium of the National Academy of Sciences, “In the Light of Evolution IX: Clonal Reproduction: Alternatives to Sex,” held January 9–10, 2015, at the Arnold and Mabel Beckman Center of the National Academies of Sciences and Engineering in Irvine, CA. The complete program and video recordings of most presentations are available on the NAS website at www.nasonline.org/ILE_IX_Clonal_Reproduction .

This article is a PNAS Direct Submission.

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